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1.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34548405

RESUMO

Animals interact with microbes that affect their performance and fitness, including endosymbionts that reside inside their cells. Maternally transmitted Wolbachia bacteria are the most common known endosymbionts, in large part because of their manipulation of host reproduction. For example, many Wolbachia cause cytoplasmic incompatibility (CI) that reduces host embryonic viability when Wolbachia-modified sperm fertilize uninfected eggs. Operons termed cifs control CI, and a single factor (cifA) rescues it, providing Wolbachia-infected females a fitness advantage. Despite CI's prevalence in nature, theory indicates that natural selection does not act to maintain CI, which varies widely in strength. Here, we investigate the genetic and functional basis of CI-strength variation observed among sister Wolbachia that infect Drosophila melanogaster subgroup hosts. We cloned, Sanger sequenced, and expressed cif repertoires from weak CI-causing wYak in Drosophila yakuba, revealing mutations suspected to weaken CI relative to model wMel in D. melanogaster A single valine-to-leucine mutation within the deubiquitylating (DUB) domain of the wYak cifB homolog (cidB) ablates a CI-like phenotype in yeast. The same mutation reduces both DUB efficiency in vitro and transgenic CI strength in the fly, each by about twofold. Our results map hypomorphic transgenic CI to reduced DUB activity and indicate that deubiquitylation is central to CI induction in cid systems. We also characterize effects of other genetic variation distinguishing wMel-like cifs Importantly, CI strength determines Wolbachia prevalence in natural systems and directly influences the efficacy of Wolbachia biocontrol strategies in transinfected mosquito systems. These approaches rely on strong CI to reduce human disease.


Assuntos
Citoplasma/patologia , Drosophila melanogaster/microbiologia , Embrião não Mamífero/microbiologia , Mutação , Simbiose , Ubiquitinação , Wolbachia/fisiologia , Animais , Citoplasma/microbiologia , Enzimas Desubiquitinantes/metabolismo , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Feminino , Masculino
2.
FASEB J ; 35(9): e21332, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34423867

RESUMO

Emerging research has highlighted the capacity of microRNA-23a-3p (miR-23a-3p) to alleviate inflammatory pain. However, the molecular mechanism by which miR-23a-3p attenuates inflammatory pain is yet to be fully understood. Hence, the current study aimed to elucidate the mechanism by which miR-23a-3p influences inflammatory pain. Bioinformatics was initially performed to predict the inflammatory pain related downstream targets of miR-23a-3p in macrophage-derived extracellular vesicles (EVs). An animal inflammatory pain model was established using Complete Freund's Adjuvant (CFA). The miR-23a-3p expression was downregulated in the microglia of CFA-induced mice, after which the inflammatory factors were determined by ELISA. FISH and immunofluorescence were performed to analyze the co-localization of miR-23a-3p and microglia. Interestingly, miR-23a-3p was transported to the microglia via M2 macrophage-EVs, which elevated the mechanical allodynia and the thermal hyperalgesia thresholds in mice model. The miR-23a-3p downstream target, USP5, was found to stabilize HDAC2 via deubiquitination to promote its expression while inhibiting the expression of NRF2. Taken together, the key findings of the current study demonstrate that macrophage-derived EVs containing miR-23a-3p regulates the HDAC2/NRF2 axis by decreasing USP5 expression to alleviate inflammatory pain, which may provide novel therapeutic targets for the treatment of inflammatory pain.


Assuntos
Vesículas Extracelulares/metabolismo , Histona Desacetilase 2/metabolismo , Inflamação/metabolismo , Macrófagos/citologia , Fator 2 Relacionado a NF-E2/metabolismo , Dor/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Enzimas Desubiquitinantes/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Estabilidade Enzimática , Vesículas Extracelulares/genética , Inflamação/genética , Inflamação/terapia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , Modelos Biológicos , Dor/genética , Manejo da Dor , Proteases Específicas de Ubiquitina/genética , Ubiquitinação
3.
J Pharm Pharm Sci ; 24: 390-399, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34319871

RESUMO

PURPOSE: SARS-CoV-2 infection is associated with substantial mortality and high morbidity. This study tested the effect of angiotensin II type I receptor blocker, losartan, on SARS-CoV-2 replication and inhibition of the papain-like protease of the virus. METHODS: The dose-dependent inhibitory effect of losartan, in concentrations from 1µM to 100µM as determined by quantitative cell analysis combining fluorescence microscopy, image processing, and cellular measurements (Cellomics analysis) on SARS-CoV-2 replication was investigated in Vero E6 cells. The impact of losartan on deubiquitination and deISGylation of SARS-CoV-2 papain-like protease (PLpro) were also evaluated.  Results: Losartan reduced PLpro cleavage of tetraUbiquitin to diUbiquitin.  It was less effective in inhibiting PLpro's cleavage of ISG15-AMC than Ubiquitin-AMC.  To determine if losartan inhibited SARS-CoV-2 replication, losartan treatment of SARS-CoV-2 infected Vero E6 was examined. Losartan treatment one hour prior to SARS-CoV-2 infection reduced levels of SARS-CoV-2 nuclear protein, an indicator of virus replication, by 80% and treatment one-hour post-infection decreased viral replication by 70%. CONCLUSION: Losartan was not an effective inhibitor of deubiquitinase or deISGylase activity of the PLpro but affected the SARS-CoV-2 replication of Vero E6 cells in vitro.  As losartan has a favorable safety profile and is currently available it has features necessary for efficacious drug repurposing and treatment of COVID-19.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Antivirais/farmacologia , Losartan/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/tratamento farmacológico , Chlorocebus aethiops , Biologia Computacional , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Ubiquitina/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos
4.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201062

RESUMO

Cancer is a disorder of cell growth and proliferation, characterized by different metabolic pathways within normal cells. The Warburg effect is a major metabolic process in cancer cells that affects the cellular responses, such as proliferation and apoptosis. Various signaling factors down/upregulate factors of the glycolysis pathway in cancer cells, and these signaling factors are ubiquitinated/deubiquitinated via the ubiquitin-proteasome system (UPS). Depending on the target protein, DUBs act as both an oncoprotein and a tumor suppressor. Since the degradation of tumor suppressors and stabilization of oncoproteins by either negative regulation by E3 ligases or positive regulation of DUBs, respectively, promote tumorigenesis, it is necessary to suppress these DUBs by applying appropriate inhibitors or small molecules. Therefore, we propose that the DUBs and their inhibitors related to the Warburg effect are potential anticancer targets.


Assuntos
Apoptose , Enzimas Desubiquitinantes/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Ubiquitinação , Efeito Warburg em Oncologia , Animais , Humanos
5.
Biomolecules ; 11(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071582

RESUMO

The urgent need for novel and effective drugs against the SARS-CoV-2 coronavirus pandemic has stimulated research worldwide. The Papain-like protease (PLpro), which is essential for viral replication, shares a similar active site structural architecture to other cysteine proteases. Here, we have used representatives of the Ovarian Tumor Domain deubiquitinase family OTUB1 and OTUB2 along with the PLpro of SARS-CoV-2 to validate and rationalize the binding of inhibitors from previous SARS-CoV candidate compounds. By forming a new chemical bond with the cysteine residue of the catalytic triad, covalent inhibitors irreversibly suppress the protein's activity. Modeling covalent inhibitor binding requires detailed knowledge about the compounds' reactivities and binding. Molecular Dynamics refinement simulations of top poses reveal detailed ligand-protein interactions and show their stability over time. The recently discovered selective OTUB2 covalent inhibitors were used to establish and validate the computational protocol. Structural parameters and ligand dynamics are in excellent agreement with the ligand-bound OTUB2 crystal structures. For SARS-CoV-2 PLpro, recent covalent peptidomimetic inhibitors were simulated and reveal that the ligand-protein interaction is very dynamic. The covalent and non-covalent docking plus subsequent MD refinement of known SARS-CoV inhibitors into DUBs and the SARS-CoV-2 PLpro point out a possible approach to target the PLpro cysteine protease from SARS-CoV-2. The results show that such an approach gives insight into ligand-protein interactions, their dynamic character, and indicates a path for selective ligand design.


Assuntos
Enzimas Desubiquitinantes/antagonistas & inibidores , Inibidores de Proteases/química , SARS-CoV-2/metabolismo , Proteases Virais/química , Sítios de Ligação , COVID-19/patologia , Domínio Catalítico , Enzimas Desubiquitinantes/metabolismo , Desenho de Fármacos , Feminino , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Inibidores de Proteases/metabolismo , SARS-CoV-2/isolamento & purificação , Proteases Virais/metabolismo
6.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34070986

RESUMO

Oral health is an integral part of the general health and well-being of individuals. The presence of oral disease is potentially indicative of a number of systemic diseases and may contribute to their early diagnosis and treatment. The ubiquitin (Ub) system has been shown to play a role in cellular immune response, cellular development, and programmed cell death. Ubiquitination is a post-translational modification that occurs in eukaryotes. Its mechanism involves a number of factors, including Ub-activating enzymes, Ub-conjugating enzymes, and Ub protein ligases. Deubiquitinating enzymes, which are proteases that reversely modify proteins by removing Ub or Ub-like molecules or remodeling Ub chains on target proteins, have recently been regarded as crucial regulators of ubiquitination-mediated degradation and are known to significantly affect cellular pathways, a number of biological processes, DNA damage response, and DNA repair pathways. Research has increasingly shown evidence of the relationship between ubiquitination, deubiquitination, and oral disease. This review investigates recent progress in discoveries in diseased oral sites and discusses the roles of ubiquitination and deubiquitination in oral disease.


Assuntos
Doenças da Boca/metabolismo , Processamento de Proteína Pós-Traducional , Doenças Dentárias/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Síndrome de Dente Quebrado/metabolismo , Cárie Dentária/metabolismo , Sensibilidade da Dentina/metabolismo , Enzimas Desubiquitinantes/metabolismo , Previsões , Gengivite/metabolismo , Humanos , Neoplasias Bucais/metabolismo , Proteínas de Neoplasias/metabolismo , Doenças Periodontais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo
7.
Nat Commun ; 12(1): 3497, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108453

RESUMO

Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.


Assuntos
Histonas/metabolismo , Ubiquitinação/fisiologia , Cromatina/química , Cromatina/metabolismo , Enzimas Desubiquitinantes/metabolismo , Epigênese Genética , Histonas/química , Humanos , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Sirtuína 1/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo
8.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070420

RESUMO

Oct4 is an important mammalian POU family transcription factor expressed by early human embryonic stem cells (hESCs). The precise level of Oct4 governs the pluripotency and fate determination of hESCs. Several post-translational modifications (PTMs) of Oct4 including phosphorylation, ubiquitination, and SUMOylation have been reported to regulate its critical functions in hESCs. Ubiquitination and deubiquitination of Oct4 should be well balanced to maintain the pluripotency of hESCs. The protein turnover of Oct4 is regulated by several E3 ligases through ubiquitin-mediated degradation. However, reversal of ubiquitination by deubiquitinating enzymes (DUBs) has not been reported for Oct4. In this study, we generated a ubiquitin-specific protease 3 (USP3) gene knockout using the CRISPR/Cas9 system and demonstrated that USP3 acts as a protein stabilizer of Oct4 by deubiquitinating Oct4. USP3 interacts with endogenous Oct4 and co-localizes in the nucleus of hESCs. The depletion of USP3 leads to a decrease in Oct4 protein level and loss of pluripotent morphology in hESCs. Thus, our results show that USP3 plays an important role in controlling optimum protein level of Oct4 to retain pluripotency of hESCs.


Assuntos
Carcinoma Embrionário/genética , Enzimas Desubiquitinantes/metabolismo , Células-Tronco Embrionárias/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Técnicas de Inativação de Genes , Humanos , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Análise de Célula Única , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética
9.
Nat Commun ; 12(1): 4033, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188037

RESUMO

In response to DNA double-strand breaks (DSBs), repair proteins are recruited to the damaged sites. Ubiquitin signaling plays a critical role in coordinating protein recruitment during the DNA damage response. Here, we find that the microRNA biogenesis factor DGCR8 promotes tumor resistance to X-ray radiation independently of its Drosha-binding ability. Upon radiation, the kinase ATM and the deubiquitinase USP51 mediate the activation and stabilization of DGCR8 through phosphorylation and deubiquitination. Specifically, radiation-induced ATM-dependent phosphorylation of DGCR8 at serine 677 facilitates USP51 to bind, deubiquitinate, and stabilize DGCR8, which leads to the recruitment of DGCR8 and DGCR8's binding partner RNF168 to MDC1 and RNF8 at DSBs. This, in turn, promotes ubiquitination of histone H2A, repair of DSBs, and radioresistance. Altogether, these findings reveal the non-canonical function of DGCR8 in DSB repair and suggest that radiation treatment may result in therapy-induced tumor radioresistance through ATM- and USP51-mediated activation and upregulation of DGCR8.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a RNA/metabolismo , Tolerância a Radiação/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Enzimas Desubiquitinantes/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Histonas/metabolismo , Humanos , Células MCF-7 , Neoplasias/genética , Neoplasias/radioterapia , Fosforilação , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/metabolismo
10.
Biochem Biophys Res Commun ; 562: 94-99, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34049206

RESUMO

Conjugation of K48-linked ubiquitin chains to intracellular proteins mainly functions as a signal for proteasomal degradation. The conjugating enzyme E2-25K synthesizes not only canonical (noncyclic) but also cyclic K48-linked ubiquitin chains. Although the cyclic conformation is expected to repress molecular recognition by ubiquitin binding proteins due to restricting the flexibility of the ubiquitin subunits in a chain, multiple proteins are reported to associate with cyclic ubiquitin chains similar to noncyclic chains. However, the molecular mechanism of how cyclic ubiquitin chains are recognized remains unclear. Here we investigated the effect of cyclization on ubiquitin-chain cleavage and molecular recognition by a K48-linkage specific deubiquitinating enzyme OTUB1 for cyclic diubiquitin by NMR spectroscopic analyses. Compared to noncyclic diubiquitin, we observed slow but unambiguously detectable cleavage of cyclic diubiquitin to monoubiquitin by OTUB1. Intriguingly, upon ubiquitin chain cleavage, cyclic diubiquitin appeared to alter its "autoinhibited" conformation to an incompletely but partially accessible conformation, induced by interaction with OTUB1 via the ubiquitin-subunit specific recognition patches and adjacent surfaces. These data imply that cyclic ubiquitin chains may exist stably in cells in spite of the presence of deubiquitinating enzymes and that these chains can be recognized by intracellular proteins in a manner distinct from that of noncyclic ubiquitin chains.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Lisina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Ciclização , Humanos , Cinética , Isótopos de Nitrogênio , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Ubiquitina/química
11.
Arch Virol ; 166(8): 2173-2185, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34037855

RESUMO

Infectious bronchitis virus (IBV) is the only coronavirus known to infect poultry. The replication and pathogenesis of IBV are poorly understood, mainly because of the unavailability of a robust cell culture system. Here, we report that an active ubiquitin proteasome system (UPS) is necessary for efficient replication of IBV in Vero cells. Synthesis of IBV-specific RNA as well as viral protein is hampered in the presence of chemical inhibitors specific for the UPS. Like other coronaviruses, IBV encodes a papain-like protease (PLpro) that exhibits in vitro deubiquitinase activity in addition to proteolytically processing the replicase polyprotein. Our results show that the IBV PLpro enzyme inhibits the synthesis of interferon beta (IFNß) in infected chicken embryonic fibroblast (DF-1) cells and that this activity is enhanced in the presence of melanoma differentiation-associated protein 5 (MDA5) and TANK binding kinase 1 (TBK1). IBV PLpro, when overexpressed in DF-1 cells, deubiquitinates MDA5 and TBK1. Both of these proteins, along with other adapter molecules such as MAVS, IKKε, and IRF3, form a signaling cascade for the synthesis of IFNß. Ubiquitination of MDA5 and TBK1 is essential for their activation, and their deubiquitination by IBV PLpro renders them unable to participate in antiviral signaling. This study shows for the first time that there is cross-talk between the UPS and the innate immune response during IBV infection and that the deubiquitinase activity of IBV PLpro is involved in its activity as an IFN antagonist. This insight will be useful for designing better antivirals targeting the catalytic activity of the IBV PLpro enzyme.


Assuntos
Imunidade Inata , Vírus da Bronquite Infecciosa/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Replicação Viral , Animais , Linhagem Celular , Galinhas , Chlorocebus aethiops , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Enzimas Desubiquitinantes/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Células Vero
12.
J Cell Biol ; 220(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33792613

RESUMO

Numerous proteins that have hydrophobic transmembrane domains (TMDs) traverse the cytosol and posttranslationally insert into cellular membranes. It is unclear how these hydrophobic membrane proteins evade recognition by the cytosolic protein quality control (PQC), which typically recognizes exposed hydrophobicity in misfolded proteins and marks them for proteasomal degradation by adding ubiquitin chains. Here, we find that tail-anchored (TA) proteins, a vital class of membrane proteins, are recognized by cytosolic PQC and are ubiquitinated as soon as they are synthesized in cells. Surprisingly, the ubiquitinated TA proteins are not routed for proteasomal degradation but instead are handed over to the targeting factor, TRC40, and delivered to the ER for insertion. The ER-associated deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins after their insertion into the ER. Thus, our data suggest that deubiquitinases rescue posttranslationally targeted membrane proteins that are inappropriately ubiquitinated by PQC in the cytosol.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina Tiolesterase/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Domínios Proteicos/fisiologia , Transporte Proteico/fisiologia , Ubiquitina/metabolismo
13.
Int J Mol Sci ; 22(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919439

RESUMO

The mechanisms and physiological implications of regulated cell death (RCD) have been extensively studied. Among the regulatory mechanisms of RCD, ubiquitination and deubiquitination enable post-translational regulation of signaling by modulating substrate degradation and signal transduction. Deubiquitinases (DUBs) are involved in diverse molecular pathways of RCD. Some DUBs modulate multiple modalities of RCD by regulating various substrates and are powerful regulators of cell fate. However, the therapeutic targeting of DUB is limited, as the physiological consequences of modulating DUBs cannot be predicted. In this review, the mechanisms of DUBs that regulate multiple types of RCD are summarized. This comprehensive summary aims to improve our understanding of the complex DUB/RCD regulatory axis comprising various molecular mechanisms for diverse physiological processes. Additionally, this review will enable the understanding of the advantages of therapeutic targeting of DUBs and developing strategies to overcome the side effects associated with the therapeutic applications of DUB modulators.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Morte Celular Regulada , Ubiquitina/metabolismo , Animais , Humanos , Ubiquitinação
14.
Int J Mol Sci ; 22(9)2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922750

RESUMO

The ubiquitin (Ub) proteasome system (UPS) plays a pivotal role in regulation of numerous cellular processes, including innate and adaptive immune responses that are essential for restriction of the virus life cycle in the infected cells. Deubiquitination by the deubiquitinating enzyme, deubiquitinase (DUB), is a reversible molecular process to remove Ub or Ub chains from the target proteins. Deubiquitination is an integral strategy within the UPS in regulating survival and proliferation of the infecting virus and the virus-invaded cells. Many viruses in the infected cells are reported to encode viral DUB, and these vial DUBs actively disrupt cellular Ub-dependent processes to suppress host antiviral immune response, enhancing virus replication and thus proliferation. This review surveys the types of DUBs encoded by different viruses and their molecular processes for how the infecting viruses take advantage of the DUB system to evade the host immune response and expedite their replication.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Viroses/imunologia , Vírus/enzimologia , Animais , Enzimas Desubiquitinantes/química , Humanos , Evasão da Resposta Imune , Estágios do Ciclo de Vida , Ubiquitinação , Proteínas Virais/química , Viroses/enzimologia , Viroses/virologia , Replicação Viral , Vírus/imunologia
15.
J Immunol ; 206(8): 1832-1843, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33789983

RESUMO

CARD9 is an essential adaptor protein in antifungal innate immunity mediated by C-type lectin receptors. The activity of CARD9 is critically regulated by ubiquitination; however, the deubiquitinases involved in CARD9 regulation remain incompletely understood. In this study, we identified ovarian tumor deubiquitinase 1 (OTUD1) as an essential regulator of CARD9. OTUD1 directly interacted with CARD9 and cleaved polyubiquitin chains from CARD9, leading to the activation of the canonical NF-κB and MAPK pathway. OTUD1 deficiency impaired CARD9-mediated signaling and inhibited the proinflammatory cytokine production following fungal stimulation. Importantly, Otud1 -/- mice were more susceptible to fungal infection than wild-type mice in vivo. Collectively, our results identify OTUD1 as an essential regulatory component for the CARD9 signaling pathway and antifungal innate immunity through deubiquitinating CARD9.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Candida albicans/fisiologia , Candidíase/imunologia , Enzimas Desubiquitinantes/metabolismo , Neutrófilos/imunologia , Animais , Células Cultivadas , Citotoxicidade Imunológica , Enzimas Desubiquitinantes/genética , Modelos Animais de Doenças , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Células RAW 264.7 , Transdução de Sinais , Ubiquitinação
16.
J Virol ; 95(13): e0046021, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33827943

RESUMO

Ubiquitination plays an important role in human immunodeficiency virus 1 (HIV-1) infection. HIV proteins such as Vif and Vpx mediate the degradation of the host proteins APOBEC3 and SAMHD1, respectively, through the proteasome pathway. However, whether deubiquitylating enzymes play an essential role in HIV-1 infection is largely unknown. Here, we demonstrate that the deubiquitinase USP21 potently inhibits HIV-1 production by indirectly downregulating the expression of HIV-1 transactivator of transcription (Tat), which is essential for transcriptional elongation in HIV-1. USP21 deubiquitylates Tat via its deubiquitinase activity, but a stronger ability to reduce Tat expression than a dominant-negative ubiquitin mutant (Ub-KO) showed that other mechanisms may contribute to USP21-mediated inhibition of Tat. Further investigation showed that USP21 downregulates cyclin T1 mRNA levels by increasing methylation of histone K9 in the promoter of cyclin T1, a subunit of the positive transcription elongation factor b (P-TEFb) that interacts with Tat and transactivation response element (TAR) and is required for transcription stimulation and Tat stability. Moreover, USP21 had no effect on the function of other HIV-1 accessory proteins, including Vif, Vpr, Vpx, and Vpu, indicating that USP21 was specific to Tat. These findings improve our understanding of USP21-mediated functional suppression of HIV-1 production. IMPORTANCE Ubiquitination plays an essential role in viral infection. Deubiquitinating enzymes (DUBs) reverse ubiquitination by cleaving ubiquitins from target proteins, thereby affecting viral infection. The role of the members of the USP family, which comprises the largest subfamily of DUBs, is largely unknown in HIV-1 infection. Here, we screened a series of USP members and found that USP21 inhibits HIV-1 production by specifically targeting Tat but not the other HIV-1 accessory proteins. Further investigations revealed that USP21 reduces Tat expression in two ways. First, USP21 deubiquitinates polyubiquitinated Tat, causing Tat instability, and second, USP21 reduces the mRNA levels of cyclin T1 (CycT1), an important component of P-TEFb, that leads to Tat downregulation. Thus, in this study, we report a novel role of the deubiquitinase, USP21, in HIV-1 infection. USP21 represents a potentially useful target for the development of novel anti-HIV drugs.


Assuntos
Ciclina T/metabolismo , Enzimas Desubiquitinantes/metabolismo , HIV-1/crescimento & desenvolvimento , Ubiquitina Tiolesterase/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/biossíntese , Ciclina T/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Células Jurkat , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/análise , Replicação Viral/genética
17.
Mol Immunol ; 135: 28-35, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33857816

RESUMO

Type I interferon (IFN-I) plays pivotal roles in defense against viral infection. HSV-1 has evolved multiple strategies to evade IFN-I antiviral response. In this study, we revealed a new mechanism that HSV-1-encoded ICP0 regulates the host deubiquitinase BRCC36 to inhibit IFN-I antiviral response. We found that HSV-1 infection rapidly downregulates BRCC36 proteins at the early stage of infection. Further studies demonstrated that HSV-1-encoded ICP0 induces K48-linked polyubiquitination and degradation of BRCC36. Importantly, HSV-1-induced BRCC36 degradation promotes downmodulation of IFN-I receptor IFNAR1, thus restricting host IFN-I antiviral response to facilitate HSV-1 early infection. These findings uncover a novel immune evasion mechanism exploited by HSV-1 and could provide potential strategies for anti-HSV-1 therapy.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Herpesvirus Humano 1/imunologia , Proteínas Imediatamente Precoces/metabolismo , Evasão da Resposta Imune/imunologia , Interferon Tipo I/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação para Baixo , Células HEK293 , Células HeLa , Células Hep G2 , Herpes Simples/imunologia , Herpes Simples/terapia , Humanos , Interferon Tipo I/imunologia , Camundongos , Células RAW 264.7 , Receptor de Interferon alfa e beta/metabolismo , Ubiquitinação/fisiologia , Células Vero
18.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925279

RESUMO

Ubiquitylation and deubiquitylation are reversible protein post-translational modification (PTM) processes involving the regulation of protein degradation under physiological conditions. Loss of balance in this regulatory system can lead to a wide range of diseases, such as cancer and inflammation. As the main members of the deubiquitinases (DUBs) family, ubiquitin-specific peptidases (USPs) are closely related to biological processes through a variety of molecular signaling pathways, including DNA damage repair, p53 and transforming growth factor-ß (TGF-ß) pathways. Over the past decade, increasing attention has been drawn to USPs as potential targets for the development of therapeutics across diverse therapeutic areas. In this review, we summarize the crucial roles of USPs in different signaling pathways and focus on advances in the development of USP inhibitors, as well as the methods of screening and identifying USP inhibitors.


Assuntos
Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/metabolismo , Enzimas Desubiquitinantes/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteólise , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitinação/fisiologia
19.
PLoS Pathog ; 17(3): e1009403, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33735221

RESUMO

Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell's exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Humanos , Síndrome Respiratória e Reprodutiva Suína/virologia , Suínos , Montagem de Vírus/fisiologia , Replicação Viral/fisiologia
20.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669244

RESUMO

Aurora kinases are serine/threonine kinases required for cell proliferation and are overexpressed in many human cancers. Targeting Aurora kinases has been a therapeutic strategy in cancer treatment. Here, we attempted to identify a deubiquitinase (DUB) that regulates Aurora kinase A (Aurora-A) protein stability and/or kinase activity as a potential cancer therapeutic target. Through pull-down assays with the human DUB library, we identified OTUD6A as an Aurora-A-specific DUB. OTUD6A interacts with Aurora-A through OTU and kinase domains, respectively, and deubiquitinates Aurora-A. Notably, OTUD6A promotes the protein half-life of Aurora-A and activates Aurora-A by increasing phosphorylation at threonine 288 of Aurora-A. From qPCR screening, we identified and validated that the cancer gene CKS2 encoding Cyclin-dependent kinases regulatory subunit 2 is the most upregulated cell cycle regulator when OTUD6A is overexpressed. The results suggest that OTUD6A may serve as a therapeutic target in human cancers.


Assuntos
Aurora Quinase A/metabolismo , Enzimas Desubiquitinantes/metabolismo , Aurora Quinase A/genética , Quinases relacionadas a CDC2 e CDC28/genética , Proteínas de Ciclo Celular/genética , Enzimas Desubiquitinantes/genética , Expressão Gênica , Células HEK293 , Meia-Vida , Humanos , Fases de Leitura Aberta , Fosforilação , Estabilidade Proteica , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Genética/genética , Transfecção
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